A Streptomyces strain antagonistic to Fusarium graminearum and its application

Abstract

The present invention provides a strain of Streptomyces spectabilis FJL123, belonging to the technical field of biological control of plant diseases. The Streptomyces FJL123 was deposited at the China Center for Type Culture Collection on September 12, 2023. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20231691. The Streptomyces spectabilis FJL123 of the present invention has a growth inhibition rate of over 98% against Fusarium graminearum FG1, and the fermentation broth of Streptomyces FJL123 significantly reduces the wheat diseases caused by Fusarium graminearum and the disease severity, and the control effect reaches 53.34%.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control of plant diseases, and particularly relates to a Streptomyces strain antagonistic to Fusarium graminearum and its application. Background Art

[0002] Fusarium head blight of wheat is a wheat disease caused by the infection of wheat crops by a complex population of Fusarium spp. The main pathogenic fungi of Fusarium head blight of wheat are mainly Fusarium graminearum, Fusarium moniliforme, Fusarium culmorum, Fusarium avenaceum, Fusarium asiaticum, etc. Among them, Fusarium graminearum and Fusarium asiaticum are the main pathogenic fungi of Fusarium head blight of wheat in China.

[0003] At present, the control measures for Fusarium head blight of wheat mainly include: agricultural basic control, resistance breeding, chemical control, biological control, etc. Among them, chemical control is the control measure that has been used for the longest time in history, and ecological and sustainable biological control, as well as the most fundamental and effective resistance breeding, have also received increasing attention. Biological control refers to a means of controlling plant diseases by relying on living organisms such as animals, plants, and microorganisms or their secondary metabolites produced. The mechanisms by which antagonistic bacteria play biological control mainly include the production of antibiotics with antibiotic effects, competitive effects of nutrients and space, bacteriolysis of dissolving pathogenic bacteria cells through active substances or the bacteria themselves in metabolism, and induction of systemic resistance in plants. For antagonistic bacteria, they act on the targets of pathogenic bacteria cells, such as cell walls, cell membranes, etc., by secreting active antibacterial substances, interfering with the normal functions of the protein synthesis system and energy metabolism system of pathogenic bacteria cells, and then inhibiting or killing pathogenic bacteria.

[0004] Among many research reports on the biological control of Fusarium head blight of wheat, some active compounds with antagonistic effects against the pathogen of Fusarium head blight of wheat have been discovered.For example, Xu Li, Lu Changying, etc. isolated the antibacterial protein N1235 with antagonistic activity against Fusarium graminearum from antagonistic bacteria (Lu Changying, Ji Mingdong, Li Peiyuan, Jia Xiaokui. Determination of the antibacterial activity of antibacterial protein "N1235" against Fusarium graminearum and its efficacy test [J]. Agricultural Environment and Development, 2000, (01): 23-24.; Xu Li, Chen Xiaojie, Cao Jingting, Liu Chuchu, Ding Ting, Jiang Teng. Disease resistance mechanism of biocontrol bacteria DZSG23 against Fusarium graminearum [J]. Acta Agriculturae Zhejiangensis, 2020, 32(11): 2001-2008.); Pei Tao, etc. isolated a macromolecular antibacterial protein with the characteristics of high temperature resistance and acid resistance from Bacillus subtilis P72, and this protein has a strong inhibitory effect on the growth and reproduction of Fusarium graminearum (Pei Tao, Ren Daming, Shi Jiao. Isolation, purification and properties of antibacterial substances from antagonistic bacteria P72 against Fusarium graminearum [J]. Journal of Anhui Agricultural Sciences, 2009, 37(06): 2576-2577+2597.); Chen Hong, etc. found that Bacillus sp. X2-23 has a strong inhibitory effect on various diseases such as Fusarium graminearum and Rhizoctonia solani of rice (Chen Hong, Li Ping, Gui Yao, Wang Shiquan, Wang Lingxia. Identification of chitinase-producing bacteria X2-23 that inhibits multiple plant pathogens [J]. Journal of Sichuan University (Natural Science Edition), 2002(S1): 45-49.); Cheng Chao found that the antibacterial active substance HSAF produced by Lysobacter enzymogenes OH11 has a strong inhibitory effect on Fusarium graminearum, Valsa mali, Magnaporthe oryzae and Sclerotinia sclerotiorum of rapeseed, etc. (Cheng Chao. Preliminary study on the action mechanism of antibacterial active substance HSAF of Lysobacter enzymogenes against Fusarium graminearum [D]. Nanjing Agricultural University, 2016.); El-Shatoury found that the crude extracts of metabolites of two strains of actinomycetes, MG788011 and MG788012, not only prevent the occurrence of chocolate spot disease spots on broad beans, but also promote the growth of broad bean plants (El-Shatoury SA, Ameen F, Moussa H, Abdul Wahid O, Dewedar A, AlNadhari S. Biocontrol of chocolate spot disease (Botrytis cinerea) in faba bean using endophytic actinomycetes Streptomyces: a field study to compare application techniques [J]. PeerJ. 2020, 8(3) e8582.); However, there is no research on the related effects of actinomycetes on the pathogen Fusarium graminearum of Fusarium head blight. Therefore, there is an urgent need to provide an actinomycete that can antagonize Fusarium graminearum and has a good control effect on Fusarium head blight. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a strain of Streptomyces that antagonizes Fusarium graminearum, and this strain can effectively control wheat scab.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a strain of Streptomyces spectabilis FJL123, which was deposited at the China Center for Type Culture Collection, Wuhan University on September 12, 2023, and the deposit number is: CCTCC M 20231691.

[0008] The present invention also provides an application of the above-mentioned Streptomyces spectabilis FJL123 in inhibiting Fusarium graminearum.

[0009] The present invention also provides an application of the above-mentioned Streptomyces spectabilis FJL123 in controlling plant diseases.

[0010] The present invention also provides an application of the above-mentioned Streptomyces spectabilis FJL123 in the preparation of products for controlling plant diseases.

[0011] Preferably, the plant disease is wheat scab.

[0012] Preferably, the product includes one or several of fermentation cultures, bio-fertilizers or drugs.

[0013] Preferably, the fermentation culture includes fermentation broth.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention takes the isolated Fusarium graminearum as the research object, screens out Streptomyces FJL123 with significant antagonistic activity against Fusarium graminearum, and systematically studies the antagonistic activity of Streptomyces FJL123 against the mycelial growth and spore germination of Fusarium graminearum. In confrontation culture, double-layer medium culture and fermentation broth activity culture, the growth inhibition rate of Fusarium graminearum FG1 is up to more than 98%, and the fermentation broth of Streptomyces FJL123 greatly reduces the wheat diseases and disease severity caused by Fusarium graminearum, and the control effect reaches 53.34%. Description of the Drawings

[0016] Figure 1It is the confrontation culture screening results of antagonistic actinomycetes against Fusarium graminearum FG1 (A: FJL123; B: FJL142; C: FJL193; D: FJL245; E: FJL266; F: FJL300; CK: control);

[0017] Figure 2 It is the determination results of the activity of antagonistic actinomycetes against Fusarium graminearum FG1 cultured on a double-layer medium (A: FJL123; B: FJL142; C: FJL193; D: FJL245; E: FJL266; F: FJL300; CK: control);

[0018] Figure 3 It is the growth status of actinomycete FJL123 on the IPS1-7 medium (1: IPS1, 2: IPS2, 3: IPS3, 4: IPS4, 5: IPS5, 6: IPS6, 7: IPS7);

[0019] Figure 4 It is the phylogenetic tree of FJL123;

[0020] Figure 5 It is the effect of the fermentation broth of FJL123 on the mycelial growth of FG1 (A: sterile water control, B: treatment with the fermentation broth of FJL12);

[0021] Figure 6 It is the effect of the fermentation broth of FJL123 on the spore germination of FG1 (A: normal ungerminated spores, B: treatment with the fermentation broth of FJL123 strain, C: treatment with diluted carbendazim solution, D: sterile control);

[0022] Figure 7 It is the effect of the fermentation broth of FJL123 on the growth of wheat indoors (A: co-culture of wheat with actinomycetes and FG1, B: co-culture of wheat with actinomycetes, C: co-culture of wheat with FG1, D: wheat cultured alone, E: co-culture of wheat with FG1 and carbendazim);

[0023] Figure 8 It is the bar chart of the stem height, root length, dry weight and disease index of wheat indoors in each treatment group.

[0024] Biological deposit description

[0025] The Streptomyces spectabilis FJL123 provided by the present invention is deposited in the China Center for Type Culture Collection of Wuhan University, with the deposit number: CCTCC M 20231691, the deposit time is September 12, 2023, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Detailed implementation manners

[0026] The present invention provides a strain of Streptomyces spectabilis FJL123, which was deposited at the China Center for Type Culture Collection, Wuhan University on September 12, 2023, with the deposit number: CCTCC M 20231691; the deposit address of the China Center for Type Culture Collection, Wuhan University is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0027] In the present invention, the mycelia of the Streptomyces spectabilis FJL123 are orange-red, with well-developed branched mycelia, and the spores are mainly oval.

[0028] The present invention also provides an application of the Streptomyces spectabilis FJL123 in inhibiting Fusarium graminearum.

[0029] The present invention also provides an application of the Streptomyces spectabilis FJL123 in preventing and controlling plant diseases. In the present invention, the plant disease is wheat scab.

[0030] The present invention also provides an application of the Streptomyces spectabilis FJL123 in preparing products for preventing and controlling plant diseases. In the present invention, the products include one or several of fermentation cultures, bio-bacterial fertilizers or drugs. In the present invention, the fermentation culture includes fermentation broth; the bio-bacterial fertilizer includes bio-bacterial agents or bio-organic fertilizers; the drug is a pesticide.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] Isolation and purification of actinomycetes:

[0034] (1) Pouring the culture medium onto plates: Heat the prepared Gao's No. 1 medium (NaCl 0.5 g / L, KNO3 1 g / L, FeSO4·7H2O 0.01 g / L, soluble starch 20 g / L, K2HSO4 0.5 g / L, agar 20 g / L, MgSO4·7H2O 0.5 g / L, pH 7.2 - 7.4. Sterilize at 121 °C for 30 min.) in a microwave oven until melted. When the temperature drops to about 50 °C, add potassium dichromate solution (the final concentration of potassium dichromate solution is 50 μg / mL), and quickly pour the mixed culture medium solution into sterile culture dishes, 15 mL per dish.

[0035] (2) Process the sample and prepare gradient dilution solutions: Weigh 3 g of soil and place it in a sterile mortar. Add 1 mL of sterile water and grind thoroughly. Take 100 μL of the ground solution and add it to a centrifuge tube containing 900 μL of sterile water, and mix well. Prepare solutions with different dilution factors of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 in this way.

[0036] (3) Coating: Use a pipette to separately aspirate 100 μL of solutions with different dilution factors and drop them onto the surface of the culture medium. Then use a sterile spreading rod to spread evenly. In a sterile operating bench, let it stand for 10 min to allow the coating solution to be fully absorbed by the culture medium. Repeat this 3 times.

[0037] (4) Cultivation: After coating, seal the petri dish with a sealing film and place the petri dish upside down in an incubator at a constant temperature of 28 °C for cultivation.

[0038] (5) Isolation and purification: During the cultivation process, pay attention to observing the growth of colonies. When actinomycete colonies appear, use a sterile inoculation loop to transfer the colonies to a new petri dish containing Gause's No. 1 solid culture medium and cultivate at a constant temperature of 28 °C. Repeat this process multiple times until each petri dish contains only one type of actinomycete and no other contaminants.

[0039] (6) Strain preservation: Pick the actinomycete cells (visible to the naked eye) into a 2 mL sterile cryopreservation tube, add 1.5 mL of 20% sterilized glycerol, and mix well with a pipette. Store it in an ultra-low temperature freezer at -80 °C.

[0040] Example 2

[0041] Screening of Fusarium graminearum - antagonistic actinomycetes: Screen strains with antagonistic activity through the plate confrontation method and the double - layer culture medium method.

[0042] a. Plate confrontation method

[0043] Judge the antagonistic activity of the strain by co - culturing the actinomycetes isolated in Example 1 with the isolated Fusarium graminearum. That is, in a petri dish containing PDA medium (200 g / L of potato, 20 g / L of glucose, 18 g / L of agar powder. Sterilize at 121 °C for 30 min), inoculate the actinomycetes isolated in Example 1 on one side by streaking (the control group does not inoculate actinomycetes), and inoculate a pathogen cake on the other side. Place it in an incubator at a constant temperature of 25 °C for cultivation. Observe the growth status of the pathogen mycelium every day. After the CK is fully grown, judge the activity of the strain according to the growth of the mycelium and the size of the inhibition zone in the co - culture petri dish.

[0044] b. Double - layer culture medium method

[0045] Prepare Petri dishes containing double-layer PDA medium in a sterile workbench. Inoculate actinomycetes on the upper layer of the medium (the control group is not inoculated). The inoculated actinomycetes need to be inoculated into ISP2 liquid medium in advance, and cultured on a shaker at 28 °C and 180 r / min for 2 days. After the culture is completed, grind thoroughly using a grinder in a sterile environment, and finally adjust the concentration to about 10 4 CFU·mL -1 , extract 100 μL and drop it onto the upper layer of the medium, and spread it evenly using a sterile spreader. After inoculation, place it in an incubator at 25 °C for 10 days. Take it out, remove the upper layer in a sterile environment, and then inoculate a 5-mm pathogen cake in the center of the lower layer of the medium, and place it in an incubator at 25 °C for culture. Observe the hyphal growth status. After the CK fills the Petri dish, screen out the active strains and calculate the inhibition rate.

[0046]

[0047] Experimental results: As shown in Table 1 and Figure 1 、 2 shown.

[0048] Table 1 Screening results of antagonistic actinomycetes against Fusarium graminearum FG1

[0049] Strain Antagonistic activity in confrontation culture Antagonistic activity in double-layer culture FJL123 98.31％ 99.76％ FJL142 41.17％ 92.46％ FJL193 43.52％ 99.16％ FJL245 47.64％ 98.38％ FJL266 40.04％ 99.84％ FJL300 42.68％ 82.67％

[0050] From Table 1 and Figure 1 it can be seen that the confrontation culture results of the isolated actinomycetes and the pathogen FG1 show that: 6 actinomycetes have good antagonistic activity against FG1, namely FJL123, FJL142, FJL193, FJL245, FJL266, FJL300. Among them, FJL123 has the highest antagonistic activity, and the inhibition of FG1 hyphal growth reaches more than 98%; the inhibition effects of the other 5 actinomycetes on FG1 hyphal growth are all between 40% and 60%.

[0051] From Table 1 and Figure 2 it can be seen that for the 6 actinomycetes with obvious inhibitory effects on the hyphal growth of Fusarium graminearum FG1 cultured in a double-layer medium, the activity results show that: the inhibition rates of actinomycetes FJL123, FJL193, FJL245, and FJL266 on FG1 hyphae are all close to 100%; the inhibition rate of FJL193 on hyphae is greater than 95%; the inhibition rate of FJL300 on FG1 hyphae is greater than 85%.

[0052] Example 3

[0053] Morphological identification of FJL123:

[0054] The compositions of ISP1-ISP7 liquid media are shown in Tables 2-8, and the preparation methods are as follows:

[0055] Table 2 Composition of ISP1 Liquid Medium

[0056] Peptone 5g Yeast extract 3g Agar (optional addition) 15g pH 7.0±0.2

[0057] Preparation method: Add the above components to 1 L of deionized water and sterilize at 121 °C for 15 min.

[0058] Table 3 Composition of ISP2 Liquid Medium

[0059] Malt extract 10g Yeast extract 4g D-Glucose 4g Agar 20g pH 7.2±0.2

[0060] Preparation method: Add the above components to 1 L of deionized water and sterilize at 121 °C for 15 min.

[0061] Table 4 Composition of ISP3 Liquid Medium

[0062] Oatmeal 20g Agar 18g Ferrous sulfate heptahydrate 0.001g Manganese chloride tetrahydrate 0.001g Zinc sulfate heptahydrate 0.001g pH 7.3±0.2

[0063] Preparation method: Add the above components to 1 L of deionized water and sterilize at 121 °C for 15 min.

[0064] Table 5 Composition of ISP4 Liquid Medium

[0065]

[0066]

[0067] Preparation method: Add the above components to 1 L of deionized water, sterilize at 121 °C for 15 min; after shaking well, pour the plate to evenly disperse the insoluble part into each petri dish.

[0068] Table 6 Composition of ISP5 Liquid Medium

[0069] L-Asparagine 1g Dipotassium hydrogen phosphate 1g Ferrous sulfate heptahydrate 0.001g Manganese chloride tetrahydrate 0.001g Zinc sulfate heptahydrate 0.001g Agar 20g pH 7.4±0.2

[0070] Preparation method: Weigh the above components, add 10 mL of glycerol and 1 L of deionized water, heat to boiling until completely dissolved; sterilize at 121 °C for 15 min; after cooling to below 55 °C, pour the plate, and pour 20 mL of the medium into each 90-mm petri dish.

[0071] Table 7 Composition of ISP6 Liquid Medium

[0072]

[0073]

[0074] Preparation method: Weigh the above components, 10 mL of glycerol, add 1 L of deionized water, heat to boiling until completely dissolved; sterilize at 121 °C for 15 min; pour plates after cooling to below 55 °C, and pour 20 mL of the medium into each 90-mm petri dish.

[0075] Table 8 Components of ISP7 Liquid Medium

[0076] L-Asparagine 1g L-Tyrosine 0.5g Dipotassium hydrogen phosphate 0.5g Magnesium sulfate heptahydrate 0.5g Sodium chloride 0.5g Ho-Le trace element solution (mL) 1 mL Agar 20g Glycerol (mL) 15g pH 7.3±0.1

[0077] The formula of 1 L of trace element solution Ho-Le is as follows: 2.85 g of boric acid, 1.8 g of manganese chloride tetrahydrate, 1.36 g of ferrous sulfate heptahydrate, 1.77 g of sodium tartrate, 26.9 mg of copper chloride dihydrate, 20.8 mg of zinc chloride, 40.4 mg of cobalt chloride hexahydrate, 25.2 mg of sodium molybdate dihydrate.

[0078] Preparation method: Weigh the above components, 15 mL of glycerol, add 1 L of deionized water, heat to boiling until completely dissolved; sterilize at 121 °C for 15 min; pour plates after cooling to below 55 °C, and pour 20 mL of the medium into each 90-mm petri dish.

[0079] Inoculate the screened strain FJL123 into ISP1 - ISP7 liquid media respectively, place it on a shaker at 28 °C and 180 r / min for 2 d. After the cultivation is completed, take 1 mL of the bacterial liquid and put it into a 2-mL sterile EP tube, and grind it thoroughly using a grinder in a sterile environment. Take 30 μL and drop it onto a plate containing Gause's No. 1 medium, and spread it evenly using a sterile spreader. Then, insert a pre-sterilized glass slide obliquely on the medium. After inoculation, place it in an incubator at 28 °C for 7 d. Observe the colony morphology, aerial mycelium and spore morphology, and record them.

[0080] The experimental results are as Figure 3 shown. It can be Figure 3 seen that when the strain FJL123 is cultured on different ISP1 - ISP7 media, there are significant differences in morphological characteristics. On ISP1 medium, the strain grows well, sporulates well, produces less pigment, the area around the colony is light dark green, the center of the colony is light dark green and white, and the texture is hard; on ISP2 medium, the strain grows well, sporulates well, and produces light red pigment; on ISP3 medium, the strain grows poorly, does not sporulate, and has weak pigment-producing ability; on ISP4 medium, the strain grows well, sporulates less, has well-developed aerial mycelium, and produces abundant red pigment; on ISP5 medium, the strain grows well, sporulates well, has well-developed aerial mycelium, and produces orange pigment; on ISP6 medium, the strain grows poorly, does not sporulate, and produces yellow pigment; on ISP7 medium, the strain grows well, sporulates well, and does not produce pigment.

[0081] Experimental Example 4

[0082] Physiological and biochemical identification of FJL123: Refer to the Manual for Systematic Identification of Common Bacteria for physiological and biochemical identification of FJL123.

[0083] (1) Esculin hydrolysis test: Pour the prepared esculin liquid medium into a test tube, sterilize it at 121 °C for 30 min and then inoculate. The blank control group is the esculin liquid medium without inoculation. Incubate it in an incubator at 28 °C for 15 d. Observe carefully. If the color of the medium becomes darker, the reaction is positive; otherwise, it is negative.

[0084] (2) Gelatin liquefaction test: Gelatin is in a liquid state above 30 °C and in a solid state below 20 °C. The purpose of this test is to observe whether the strain has the ability to produce protease. Pour the prepared medium into a test tube, sterilize it at 121 °C for 30 min and then inoculate. The blank control group is the gelatin medium without inoculating the strain. Incubate it in an incubator at 28 °C and observe its liquefaction at different times (7, 14, 21, 28 d). If there is flowing liquid below 20 °C, the reaction is positive; otherwise, it is negative.

[0085] (3) Starch hydrolysis test: Inoculate and culture the strain by the streak plate method until the strain grows well. Drop iodine solution around it. If the strain can produce amylase, a colorless and transparent band will appear around the strain, and the reaction is positive; otherwise, blue appears around the colony, and the reaction is negative.

[0086] (4) H2S production test: Pour the sterilized Czapak medium into a petri dish and inoculate the strain on the medium by the streak plate method. The blank control group is the Czapak medium without inoculating the strain. Incubate it in an incubator (28 °C) for 8 d. Pay attention to the color change of the strain and its surroundings. If there is a blackening phenomenon, it indicates that H2S is produced, and the reaction is positive; otherwise, the reaction is negative.

[0087] (5) Melanin production test: Pour the sterilized tyrosine and ISP6 media into petri dishes and inoculate the strain onto the plates by the streak plate method. The blank control group is the two media without inoculating the strain. Incubate it in an incubator (28 °C) for 8 d. Pay attention to the surroundings of the colony. If melanin is produced, it indicates that tyrosinase can be produced, and the reaction is positive. Otherwise, the reaction is negative.

[0088] (6) Catalase test: Inoculate the strain on the Gao's No. 1 solid medium by the streak plate method and culture it at 28 °C for 5 d. Prepare a 7.5% hydrogen peroxide solution and drop it onto the medium of the strain to be identified. If bubbles are produced, it indicates that the strain can produce catalase, and the reaction is positive; otherwise, it is negative.

[0089] (7) Carbon source utilization test: Sucrose, D-glucose, L-arabinose, D-fructose, D-xylose, raffinose, i-inositol, and D-mannitol were selected as carbon sources and added at a mass concentration of 1%. The strain was inoculated into the medium supplemented with different carbon sources, and the blank control group was the basal medium. It was cultured in an incubator at 28°C for 15 days, and the growth of the strain was observed carefully.

[0090] Experimental results: As shown in Table 9 and Table 10.

[0091] Table 9 Physiological and biochemical identification results of Actinomycetes FJL123

[0092]

[0093] Note: "+" indicates having the corresponding physiological and biochemical activity; "-" indicates having no corresponding physiological and biochemical activity.

[0094] Table 10 Carbon source utilization results of Actinomycetes FJL123

[0095]

[0096] Note: "+" indicates being able to utilize the corresponding carbon source; "-" indicates being unable to utilize the corresponding carbon source.

[0097] Experimental Example 5

[0098] Detection of 16S rDNA sequence of FJL123:

[0099] (1) DNA extraction: The specific method is as follows:

[0100] Preparation of bacterial cells: In a sterile operation bench, the wild Actinomycetes purified and stored in a -80°C refrigerator were inoculated onto a plate of Gao's No. 1 medium and cultured in a constant temperature incubator at 28°C for 7 - 10 days; an appropriate amount of Actinomycetes cells were picked from the plate of Gao's No. 1 medium with a sterile toothpick and placed in a 1.5 mL sterile EP tube for storage at -20°C for later use.

[0101] DNA extraction:

[0102] ① Add 1 mL of washing solution to the EP tube containing an appropriate amount of bacterial cells, shake on a vortex oscillator for 5 s, centrifuge at 12000 rpm for 1 min, and discard the supernatant.

[0103] ② Add 50 μL of lysis solution, shake to suspend, and place in a 600W microwave oven for 60 s.

[0104] ③ Add 400 μL of extraction solution (preheated at 65°C in advance), and shake on a vortex oscillator for 6 s.

[0105] ④ Add an equal volume of Tis saturated phenol / chloroform solution, invert vigorously up and down to mix well, let stand at room temperature until layered, then centrifuge at 12,000 rpm for 5 min.

[0106] ⑤ Transfer an appropriate amount of the supernatant to a 1.5 mL sterile EP tube, add an equal volume of isopropanol, invert vigorously up and down to mix well, perform alcohol precipitation in a -20°C refrigerator, after treatment for 20 min, centrifuge at 12,000 rpm for 5 min, and discard all the liquid.

[0107] ⑥ Add 1 mL of 70% ethanol to the precipitate, centrifuge at 12,000 rpm for 3 min, and discard all the liquid.

[0108] ⑦ Open the lid and place at room temperature until dry, then dissolve in 20 μL TE buffer. The extracted DNA is stored at -20°C for future use.

[0109] (2) PCR amplification: ① The PCR amplification primers are universal bacterial primers, and the primer names are 27-F and 1492-R.

[0110] ② The PCR amplification system is shown in Table 11.

[0111] Table 11 16S rRNA gene PCR amplification system

[0112]

[0113]

[0114]

[0115] (3) 16S rRNA gene sequence processing: The measured sequences were assembled using DNAman and then submitted to the NCBI database. Alignment of the corresponding nucleic acid sequences was performed in the Ezbiocloud nucleic acid database, homologous strain sequences were downloaded, and a phylogenetic tree (Neighbor-Join method) was constructed. Based on the tree construction results, further analysis and identification of the taxonomic status of the strain were carried out.

[0116] Experimental results: The phylogenetic tree of FJL123 is as Figure 4 shown. The identification results indicate that FJL123 belongs to Streptomyces spectabilis.

[0117] In summary: Based on the morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis of strain FJL123, combined with the "Common Bacterial System Identification Manual", it was determined that strain FJL123 belongs to Streptomyces spectabilis.

[0118] Example 6

[0119] Inhibitory test of the fermentation broth of FJL123 on the mycelial growth and spore germination of FG1:

[0120] Activation of the strain: The target strain stored in an ultra-low temperature freezer at -80°C was taken out, inoculated onto Gause's No. 1 solid medium using a sterile inoculation loop, and placed in an incubator at 28°C for 5 days to activate it.

[0121] Preparation of the fermentation broth: (1) Cultivation of the seed solution: In a laminar flow hood, a small amount of activated bacterial cells were picked using a sterile inoculation loop and inoculated into a prepared test tube containing ISP2 liquid medium. The test tube was sealed with a test tube stopper and placed on a shaker at 180 r / min and 28°C for 2 days. (2) Fermentation of the target strain: 1 mL of the seed solution was added to the prepared M2 liquid medium (0.01% tyrosine, 1.0% glucose, 2.1% MOPS, 1.0% soluble starch, 5.0% sucrose, 0.5% yeast extract powder, 0.025% K2SO4, 1.0% MgCl2·7H2O, 0.1% trace elements (0.45 g CuSO4·5H20, 1.0 g FeSO4·7H2O, 0.1 g MnSO4·4H2O, 0.1 g K2MOO4, 1.0 g ZnSO4·7H2O, 1 L deionized water), pH 7.2 - 7.4. Sterilized at 121°C for 30 min.) (in a 250 mL Erlenmeyer flask, each flask containing 100 mL of M2 liquid medium) and placed on a shaker at 180 r / min and 28°C for 7 days.

[0122] Treatment of the fermented bacterial liquid: After fermentation is completed, centrifuge at 5000 r / min for 5 min to separate the supernatant and the precipitate. Collect the supernatant, filter it with a sterile filter membrane, and collect it for standby.

[0123] Determination of the antibacterial activity of the FJL123 fermentation broth against the mycelial growth of FG1:

[0124] The antibacterial activity of the fermentation broth of the actinomycete strain was determined by the mycelial growth rate method. The prepared PDA medium was placed in a microwave oven and heated and melted thoroughly. When the temperature dropped to about 60 °C, use a pipette to draw 10 mL of the medium to make a PDA medium plate. After the plate cooled, in a laminar flow hood, draw 1 mL of the supernatant of the prepared actinomycete fermentation broth and spread it evenly on the surface of the medium. Let it dry naturally in the laminar flow hood. Use a punch with a diameter of 5 mm to punch out a well-grown pathogenic fungus FG1 mycelial cake, and inoculate it at the center position of the petri dish. Place it in an incubator at 25 °C for cultivation. When the control mycelium covers the plate, use the cross method to measure the mycelial diameter and calculate the mycelial growth inhibition rate.

[0125] Effect of the FJL123 fermentation broth on the mycelial growth of FG1:

[0126] In order to enable FG1 mycelium to grow in the PDA liquid medium containing the FJL123 fermentation broth, mix the FJL123 fermentation supernatant collected in 3.1.3.3 with the PDA liquid medium at a ratio of 1:10 to prepare a PDA liquid medium containing the FJL123 fermentation broth. The control group is to replace the supernatant with sterile water. Pour the prepared medium into a sterile petri dish, pick a small amount of well-grown FG1 mycelium and inoculate it into the medium. Place it in an incubator at 25 °C for cultivation. Pay attention to observation. After it is suitable for observation, take it out and observe it under a microscope.

[0127] Effect of the FJL123 fermentation broth on the spore germination of FG1:

[0128] Before conducting the spore germination test, spores of Fusarium graminearum FG1 were induced. Prepare 3% mung bean soup medium (weigh 30g mung beans, add 800mL water and boil until the mung beans burst; filter out the mung beans and add water to 1000mL, sterilize at 121℃ for 30min.), filter with 4 layers of filter cloth, divide the prepared mung bean soup medium into 250mL triangular bottles, 100mL per bottle, and sterilize at high temperature and high pressure in a high-pressure steam autoclave. After everything is ready, inoculate 3 FG1 bacterial cakes into each triangular bottle containing 3% mung bean soup medium on a sterile operating table, place it on a shaker at 25℃, 150r / min for 4d, and observe the spore production. After spore induction, filter with lens paper to collect spore liquid, centrifuge at 3000r / min to collect spores, wash spores with sterile water for 3 times, and dilute spores with sterile water to a concentration of 1×10 4 CFU·mL -1 The spore liquid was stored in a refrigerator at 4°C for later use.

[0129] Using the concave culture method, a pipette was used to add the spore liquid and FJL123 fermentation supernatant in a ratio of 3μL:3μL onto a sterile glass slide with a concave surface. The control group used sterile water instead of the fermentation liquid, and the fermentation liquid was replaced by a pre-prepared carbendazim solution according to the instructions. The slides were placed in a constant temperature incubator at 25°C for moisturizing culture. The spore germination was observed from the 4th hour. After the spores in the sterile water treatment group germinated well, the test samples were taken out, photographed under a microscope, and the data were recorded.

[0130] Experimental results: Figure 5 and Figure 6 shown.

[0131] Depend on Figure 5 It can be seen that under the condition of sterile water treatment, the mycelium growth and branching were normal, while FJL123 caused a significant reduction in the mycelium branching of FG1, and serious deformities appeared on the mycelium.

[0132] Depend on Figure 6 It can be seen that FJL123 has a good inhibitory effect on the germination of FG1 spores. The germination rate of spores treated with sterile water is more than 90%, the germination rate of spores treated with carbendazim solution is less than 5%, and the germination rate of spores treated with FJL123 is about 15%. Although a small amount of spores germinate in the spores treated with FJL123, the germinated hyphae grow very slowly, and even show growth stagnation.

[0133] Example 8

[0134] Effects of FJL123 fermentation liquid on indoor wheat growth and its control effect on diseases caused by FG1:

[0135] Preparation of FJL123 bacterial suspension: The preparation method was the same as that in Example 7. After cultivation, the supernatant of the FJL123 fermentation broth was collected by centrifugation, diluted 32 times, and reserved for use.

[0136] Preparation of FG1 spore suspension: Pick 3 - 4 FG1 bacterial cakes with a diameter of 0.5 cm and inoculate them into a sterilized 100 mL 3% mung bean soup medium, and place them in a shaker at 25°C and 150 r / min for cultivation. After 72 h, observe and count the spore amount under a microscope using a hemocytometer, and dilute the spore suspension to 10 4 CFU·mL -1 and place it in a refrigerator at 4°C for standby.

[0137] Dilution of carbendazim fungicide: Prepare a carbendazim (purchased from Shanxi Qixing) dilution solution according to the product instructions. Dilute 80% carbendazim wettable powder. According to 100 g / 666.7 m 2 , weigh 0.445 g of 80% carbendazim wettable powder, add water to 400 mL to prepare a primary carbendazim dilution solution, and then dilute the primary dilution solution 10 times to make a carbendazim target dilution solution for standby.

[0138] Cultivation of wheat and inoculation of pathogenic bacteria: Fill the organic nutrient soil into cultivation pots, soak it thoroughly with sterile water, then disinfect the wheat seeds with 2% sodium hypochlorite for 2 min, rinse them 4 times with sterile water, and after the seeds are dried, carry out the following treatments. Treatment 1: Plant the treated seeds in a seedling-raising pot, plant 6 wheat seeds in each pot, and inoculate 1 mL of FG1 spore suspension and 1 mL of FJL123 fermentation supernatant into each sowing pit during sowing, and cover with soil; Treatment 2: Plant the treated seeds in a seedling-raising pot, plant 6 wheat seeds in each pot, inoculate 1 mL of FJL123 supernatant, and do not inoculate FG1 spore suspension, and cover with soil; Treatment 3: Plant the treated seeds in a seedling-raising pot, plant 6 wheat seeds in each pot, and inoculate 1 mL of FG1 spore suspension into each sowing pit during sowing, and cover with soil; Treatment 4: Plant the treated seeds in a seedling-raising pot, plant 6 wheat seeds in each pot, do not do other treatments, and cover with soil; Treatment 5: Plant the treated seeds in a seedling-raising pot, plant 6 wheat seeds in each pot, and inoculate 1 mL of FG1 spore suspension and 1 mL of carbendazim dilution solution into each sowing pit during sowing, and cover with soil. Each treatment has 3 replicates and is cultured at room temperature. Observe the disease occurrence and growth conditions of wheat during the cultivation process. The disease grading is divided into 5 levels.

[0139] Level 0: No obvious symptoms;

[0140] Level 1: The sheath blight is less than 1 / 4 of the sheath length;

[0141] Level 2: The sheath blight accounts for 1 / 4 - 1 / 2 of the sheath length;

[0142] Level 3: The sheath blight accounts for 1 / 2 - 3 / 4 of the sheath length, and the infection reaches the wheat leaf sheath;

[0143] Level 4: The coleoptile is completely de - greened and rotted, and the first leaf sheath has obvious brown withering.

[0144] Level 5: The plant dies.

[0145] According to the formula, calculate the disease index and relative control efficacy:

[0146] Disease index = ∑(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest level)×100;

[0147] Relative control efficacy = (disease index of treatment three - disease index of treatment group) / disease index of treatment three × 100%.

[0148] Use SPSS 27.0 to analyze the data.

[0149] Experimental results: The effects of the fermentation broth of FJL123 on the growth of indoor wheat are shown in Table 12 and Figure 7 、 8 as follows.

[0150] Table 12 Effects of the fermentation broth of FJL123 on the growth of indoor wheat

[0151]

[0152]

[0153] From Table 12 and Figure 7 、 Figure 8It can be seen that when wheat infected by pathogenic bacteria is not effectively controlled, the stem height, root length and dry weight are significantly affected; when the pathogenic bacteria are effectively controlled, the stem height, root growth and dry weight of wheat can reach normal levels. The average stem height, root length and dry weight of the treatment of co-culturing wheat and FJL123 fermentation broth are 20.73 cm, 11.93 cm and 0.0407 g respectively, and the average stem height, root length and dry weight of the treatment of culturing wheat alone are 20.47 cm, 11.87 cm and 0.0403 g respectively. Therefore, for wheat treated with FJL123 fermentation broth, the average stem height, root length and dry weight are increased by 1.27%, 0.51% and 0.99% respectively; for the treatment of co-culturing wheat, FJL123 fermentation broth and FG1 spore solution, the average stem height, root length and dry weight are 19.67 cm, 9.87 cm and 0.0358 g respectively, and for the treatment of co-culturing wheat and FG1 spore solution, the average stem height, root length and dry weight are 11.3 cm, 6.73 cm and 0.0246 g respectively. There are significant differences in stem height, root length and dry weight between the two treatments (p < 0.01). The main reason is that in the treatment of co-culturing wheat and FG1 spore solution, wheat is infected by FG1 during the growth process, causing serious diseases and not being effectively controlled, which affects the normal growth of wheat. While in the treatment of co-culturing wheat, FJL123 fermentation broth and FG1 spore solution, the use of FJL123 fermentation broth greatly reduces the infection of FG1 on wheat, does not cause serious diseases, and has little impact on wheat growth. Therefore, FJL123 can prevent wheat seedling diseases and has no negative impact on wheat growth.

[0154] The influence results of the disease control effect of FJL123 fermentation broth on the diseases caused by FG1 are shown in Table 13 and Figure 8 as follows.

[0155] Table 13 Disease control effect of FJL123 fermentation broth on wheat diseases caused by FG1

[0156]

[0157] As shown in Table 13 and Figure 8It can be seen that Fusarium graminearum FG1 can infect wheat, resulting in varying degrees of disease on most wheat stems. Among the wheat plants infected by FG1 and showing symptoms, almost large areas of brown lesions appear on the stems. In severe cases, the infected parts of the plants rot, causing the wheat to lodge. No disease symptoms were observed in the wheat plants not inoculated with FG1. Among the wheat plants treated with the fermentation broth of FJL123 and inoculated with the spore suspension of FG1, a small number showed light brown lesions, but the diseased area was small and the disease severity was low, hardly affecting the growth of wheat, and no rotting of the diseased tissue occurred. Among the wheat plants treated with the diluted carbendazim solution and inoculated with the spore suspension of FG1, only a very small number of plants showed light brown spots on the stems, with little impact on plant growth. For the treatment of co-culturing wheat and FG1, the average disease index reached 88.89. When using the fermentation broth of FJL123, the average disease index decreased to 41.48, and there was a significant difference between the two treatments (p<0.01). For the treatment using the diluted carbendazim solution, the disease index was 33.33, showing a significant difference compared with the treatment of co-culturing wheat and FG1 (p<0.01). There was a significant difference between the use of the diluted carbendazim solution and the use of the fermentation broth of FJL123 (p<0.05). The control effect of the fermentation broth of FJL123 on wheat diseases caused by FG1 was 53.34%, and that of the diluted carbendazim solution was 62.50%. Therefore, under indoor conditions, the fermentation broth of FJL123 can significantly reduce wheat diseases caused by FG1, and its control effect is only slightly lower than that of the diluted carbendazim solution.

[0158] As can be seen from the above examples, the Streptomyces FJL123 screened in the present invention has good antagonistic activity against Fusarium graminearum, can significantly inhibit the mycelial growth and spore germination of Fusarium graminearum, and the fermentation broth of Streptomyces FJL123 can significantly reduce the wheat diseases and disease severity caused by Fusarium graminearum, showing a good control effect.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Streptomyces strain ( Streptomyces spectabilis ), FJL123, characterized in that It was deposited at the China Center for Type Culture Collection, Wuhan University on September 12, 2023, with the deposit number: CCTCC M 20231691.

2. Use of the Streptomyces ( Streptomyces spectabilis ) FJL123 in inhibiting Fusarium graminearum.

3. Use of the Streptomyces ( Streptomyces spectabilis ) FJL123 in controlling wheat scab.

4. Use of Streptomyces ( Streptomyces spectabilis ) FJL123 in the preparation of products for controlling Fusarium head blight of wheat.

5. The application according to claim 4, wherein the product comprises one or more of a fermentation culture, a biological bacterial fertilizer or a drug.

6. The application according to claim 5, wherein the fermentation culture comprises a fermentation broth.

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